RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage.
Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.
Watson
Nicholas B
NB
Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY 40202, USA.
Nelson
Eric
E
Digman
Michelle
M
Thornburg
Joshua A
JA
Alphenaar
Bruce W
BW
McGregor
W Glenn
WG
D002352:Carrier Proteins / Q000378:metabolism* / Q000528:radiation effects; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects*; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis / Q000502:physiology / Q000528:radiation effects; D009687:Nuclear Proteins / Q000378:metabolism / Q000528:radiation effects; D038761:Photobleaching / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D021381:Protein Transport / Q000528:radiation effects; D016196:S Phase / Q000528:radiation effects*; D014018:Tissue Distribution; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays / Q000009:adverse effects
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C485954:RAD18 protein, human; D044767:Ubiquitin-Protein Ligases; D004259:DNA-Directed DNA Polymerase